This invention relates to a method of producing a recombinant enzyme. More particularly, this invention relates to a method of producing water soluble enzymatically active recombinant glycine N-acyltransferase (GLYAT (E.C. 2.3.1.13)).
Detoxification of toxic metabolites by the human body is an essential physiological process. The detoxification process decreases the toxicity of several endogenous metabolites, such as steroid hormones, and exogenous toxins, which could include compounds in food or industrial chemicals.
The detoxification process is divided into three main phases. Phase I detoxification activates metabolites by adding functional groups. The activated compounds generated by phase I detoxification are often more reactive and toxic than the original metabolites, and are further processed by phase II detoxification systems. In phase II detoxification, a range of conjugation reactions serve to make the activated compounds less toxic and more soluble, for excretion in the urine and bile. Phase III detoxification involves the elimination of toxins from cells.
Organic acidemias are a group of metabolic disorders caused by dysfunctional organic acid metabolism. The deficiency of certain metabolic enzymes causes the accumulation of acids which are not normally present in high levels in the human body. There are several known organic acidemias, with methylmalonic acidemia, propionic acidemia, isovaleric acidemia, glutaric aciduria, and maple syrup urine disease being some common examples.
Isovaleric acidemia is an autosomal recessive disorder. It is caused by a deficiency of isovaleryl coenzyme A dehydrogenase. A deficiency of this enzyme results in accumulation of intermediates of leucine catabolism, including isovaleric acid, 3- and 4-hydroxyisovaleric acid, isovaleryl-carnitine and isovalerylglycine.
Isovalerylglycine is formed when isovaleric acid conjugates to glycine by glycine N-acyltransferase (GLYAT). The isovalerylglycine is less toxic than isovaleric acid, indicating that glycine conjugation is of critical importance in the treatment of isovaleric acidemia.
Urea cycle disorder is a genetic disorder caused by an enzyme deficiency in the urea cycle responsible for eliminating ammonia from the blood stream. In urea cycle disorders, nitrogen accumulates in the form of ammonia resulting in hyperammonemia which ultimately causes irreversible brain damage, coma and/or death.
A known method for enhancing glycine conjugation capacity in individuals suffering from organic acidemias is the administration of glycine supplements. Assays on liver samples have however shown that there is great variability in the glycine conjugation capacity in humans.
It is therefore evident that a means of augmenting the natural capacity for glycine conjugation would not only be beneficial to the general health of humans but may further present as an alternative therapeutic strategy for individuals affected by organic acidemias, urea cycle disorders, aminoacidurias, and exposure to some xenobiotic chemicals.
GLYAT is an enzyme responsible for the phase II detoxification of several toxic organic acids by means of conjugation to glycine. Several toxic compounds, both xenobiotic and endogenously derived metabolites, are detoxified by conjugation to glycine. In addition to GLYAT's role in the detoxification of benzoic acid, the enzyme is also important in the management of certain inborn errors of metabolism.
To date, no system for the bacterial expression and purification of an enzymatically active recombinant GLYAT has been reported.
A disadvantage associated with the lack of a system for expression of an enzymatically active recombinant GLYAT is that there is no commercially viable product currently available for directly improving the capacity of the glycine-conjugation detoxification system, particularly in the case of patients with metabolic disorders.